RESEARCH ARTICLE SUMMARY◥

NEURODEVELOPMENT

Thyroid hormone signaling specifies conesubtypes in human retinal organoidsKiara C. Eldred, Sarah E. Hadyniak, Katarzyna A. Hussey, Boris Brenerman,Ping-Wu Zhang, Xitiz Chamling, Valentin M. Sluch, Derek S. Welsbie, Samer Hattar,James Taylor, Karl Wahlin, Donald J. Zack, Robert J. Johnston Jr.*

INTRODUCTION: Cone photoreceptors in thehuman retina enable daytime, color, and high-acuity vision. The three subtypes of human conesare defined by the visual pigment that they ex-press: blue-opsin (short wavelength; S), green-opsin (medium wavelength; M), or red-opsin(long wavelength; L). Mutations that affect op-sin expression or function cause various formsof color blindness and retinal degeneration.

RATIONALE: Our current understanding ofthe vertebrate eye has been derived primarilyfrom the study ofmodel organisms.We studiedthe human retina to understand the develop-mental mechanisms that generate the mosaicof mutually exclusive cone subtypes. Specifica-tionofhumanconesoccurs ina two-stepprocess.First, a decision occurs between S versus L/Mcone fates. If theL/Mfate is chosen, a subsequentchoice is made between expression of L- or M-opsin. To determine the mechanism that con-trols the first decision between S and L/M conefates, we studied human retinal organoids de-rived from stem cells.

RESULTS: We found that human organoidsand retinas have similar distributions, gene ex-pression profiles, andmorphologies of cone sub-types.During development, S cones are specifiedfirst, followed by L/M cones. This temporalswitch from specification of S cones to genera-tion of L/M cones is controlled by thyroid hor-mone (TH) signaling. In retinal organoids thatlacked thyroid hormone receptor b (Thrb), allcones developed into the S subtype. Thrb bindswith high affinity to triiodothyronine (T3), themore active form of TH, to regulate gene ex-pression.We observed that addition of T3 earlyduring development inducedL/M fate in nearlyall cones. Thus, TH signaling through Thrb isnecessary and sufficient to induce L/M conefate and suppress S fate. THexists largely in twostates: thyroxine (T4), themost abundant circu-lating form of TH, and T3, which binds TH re-ceptorswith high affinity.Wehypothesized thatthe retina itself couldmodulate TH levels to con-trol subtype fates. We found that deiodinase 3(DIO3), an enzyme that degrades both T3 andT4, was expressed early in organoid and retina

development. Conversely, deiodinase 2 (DIO2),an enzyme that converts T4 to active T3, aswellas TH carriers and transporters, were expressedlater in development. Temporally dynamic ex-pression of TH-degrading and -activating pro-teins supports amodel in which the retina itselfcontrols TH levels, ensuring low TH signalingearly to specify S cones and high TH signalinglater in development to produce L/M cones.

CONCLUSION: Studies of model organismsand human epidemiology often generate hy-potheses about human biology that cannotbe studied in humans. Organoids provide asystem to determine the mechanisms of hu-man development, enabling direct testing ofhypotheses in developing human tissue. Our

studies identify temporalregulation of TH signalingas a mechanism that con-trols cone subtype specifica-tion in humans. Consistentwith our findings, pretermhuman infants with low

T3 and T4 have an increased incidence ofcolor vision defects. Moreover, our identifica-tion of a mechanism that generates one conesubtype while suppressing the other, coupledwith successful transplantation and incorpora-tion of stem cell–derived photoreceptors inmice, suggests that the promise of therapies totreat human diseases such as color blindness,retinitis pigmentosa, andmacular degenerationwill be achieved in the near future.▪

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The list of author affiliations is available in the full article online.*Corresponding author. Email: robertjohnston@jhu.eduCite this article as K. C. Eldred et al., Science 362, eaau6348(2018). DOI: 10.1126/science.aau6348

Temporally regulatedTH signaling specifiescone subtypes.(A) Embryonic stemcell–derived human reti-nal organoids [wild type(WT)] generate S andL/M cones. Blue, S-opsin;green, L/M-opsin.(B) Organoids that lackthyroid hormone receptorb (Thrb KO) generate all Scones. (C) Early activa-tion of TH signaling(WT + T3) specifiesnearly all L/M cones.(D) TH-degradingenzymes (such as DIO3)expressed early in devel-opment lower TH andpromote S fate, whereasTH-activating regulators(such as DIO2) expressedlater promote L/M fate.

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RESEARCH ARTICLE◥

NEURODEVELOPMENT

Thyroid hormone signaling specifies conesubtypes in human retinal organoidsKiara C. Eldred1, Sarah E. Hadyniak1, Katarzyna A. Hussey1, Boris Brenerman1,Ping-Wu Zhang2, Xitiz Chamling2, Valentin M. Sluch2, Derek S. Welsbie3, Samer Hattar4,James Taylor1,5, Karl Wahlin3, Donald J. Zack2,6,7,8, Robert J. Johnston Jr.1*

The mechanisms underlying specification of neuronal subtypes within the human nervoussystem are largely unknown. The blue (S), green (M), and red (L) cones of the retina enablehigh-acuity daytime and color vision.To determine the mechanism that controls S versus L/Mfates, we studied the differentiation of human retinal organoids. Organoids and retinas havesimilar distributions, expression profiles, and morphologies of cone subtypes. S cones arespecified first, followed by L/M cones, and thyroid hormone signaling controls this temporalswitch. Dynamic expression of thyroid hormone–degrading and –activating proteins withinthe retina ensures low signaling early to specify S cones and high signaling late to produceL/M cones. This work establishes organoids as a model for determining mechanisms ofhuman development with promising utility for therapeutics and vision repair.

Cone photoreceptors in the human retinaenable daytime, color, and high-acuity vi-sion (1). The three subtypes of human conesare defined by the visual pigment that theyexpress: blue-opsin (short wavelength; S),

green-opsin (medium wavelength; M), or red-opsin (long wavelength; L) (2). Specification ofhuman cones occurs in a two-step process. First,a decision occurs between S versus L/M cone fates(Fig. 1A). If the L/M fate is chosen, a subsequentchoice is made between expression of L- or M-opsins (3–6). Mutations that affect opsin expres-sion or function cause various forms of colorblindness and retinal degeneration (7–9). Greatprogress has been made in our understand-ing of the vertebrate eye through the study ofmodel organisms. However, little is known aboutthe developmental mechanisms that generatethe mosaic of mutually exclusive cone subtypesin the human retina. We studied the specifica-tion of human cone subtypes using human reti-nal organoids differentiated from stem cells(Fig. 1, D to K).Human retinal organoids generate photo-

receptors that respond to light (10–14). We foundthat human organoids recapitulate the specifica-tion of cone subtypes observed in the human ret-

ina, including the temporal generation of S conesfollowed by L and M cones. Moreover, we foundthat this regulation is controlled by thyroid hor-mone signaling,which is necessary and sufficientto control cone subtype fates through the nuclearhormone receptor thyroid hormone receptor b(Thrb). Expression of thyroid hormone–regulatinggenes suggests that retina-intrinsic temporal con-trol of thyroid hormone levels and activity gov-erns cone subtype specification. Whereas retinalorganoids have largely been studied for theirpromise of therapeutic applications (15), our workdemonstrates that human organoids can also beused to reveal fundamental mechanisms of hu-man development.

Specification of cone cells in organoidsrecapitulates development in thehuman retina

We compared features of cone subtypes in hu-man organoids with those of adult retinal tissue.Adult human retinas and organoids at day 200 ofdifferentiation displayed similar ratios of S to L/Mcones as indicated by expression of S- or L/M-opsins (adult, S = 13%, L/M = 87%; organoid, S =29%, L/M = 71%) (Fig. 1, B and C, and fig. S1A).The difference in the ratios is likely due to the im-maturity of the organoid at ~6 months comparedwith the terminally differentiated adult retina.We examined L/M cones with an antibody thatrecognizes both L- andM-opsin proteins becauseof their extremely high similarity. Both S and L/Mcones expressed the cone-rod-homeobox tran-scription factor (CRX), a critical transcription fac-tor for photoreceptor differentiation (Fig. 2, A andE) (16–18), indicating proper fate specification inorganoids. Additionally, cones in organoids andretinas displayed similar morphologies, with L/Mcones that had longer outer segments and widerinner segments than those of S cones (Fig. 2, B to

D and F to H) (19). The outer segments of coneswere shorter in organoids than in adult retinas,which is consistent with postnatal maturation(Fig. 2, D and H) (20). Thus, cone subtypes in hu-man retinal organoids displayed distributions,gene expression patterns, andmorphologies sim-ilar to those of cones of the human retina.We next examined the developmental dynam-

ics of cone subtype specification in organoids. Inthe human retina, S cones are generated duringfetal weeks 11 to 34 (days 77 to 238), whereas L/Mcones are specified later, during fetal weeks 14 to37 (days 98 to 259) (21, 22). We tracked the ratiosand densities of S and L/M cones in organoids bymeans of antibody staining over 360 days of dif-ferentiation. Cones expressing S-opsinwere firstobserved at day 150 (Fig. 2, I, L, and M). Thedensity of S cones leveled off at day 170 (Fig. 2M),at the time point when cones expressing L/M-opsin began to be observed (Fig. 2, J to M). Thepopulation of L/M cones increased dramaticallyuntil day 300 (Fig. 2, K toM), when they reacheda steady-state density. The 20-day differencebetween S- and L/M-opsin expression onset inretinal organoids is similar to the 20-day differ-ence observed in the appearance of S and L/Mcones in the fetal retina (21). These observationsshow a temporal switch from S cone specifica-tion to L/M cone specification during retinaldevelopment.We next conducted RNA sequencing (RNA-

seq) through 250 days of induced pluripotentstem cell (iPSC)–derived organoid development.We found that S-opsin RNA was expressed firstat day 111 and leveled off at day 160, whereas L/M-opsin RNA was expressed at day 160 and re-mained steady after day 180, which is consistentwith the timeline of photoreceptormaturation inorganoids and fetal retinas (Fig. 2N and fig. S1B).Moreover, CRX RNA and CRX protein were ex-pressed before opsins in organoids, which is sim-ilar to human development (Fig. 2N and fig. S1, Bto G) (23). Thus, human organoids recapitulatemany aspects of the developmental timeline ofcone subtype specification observed in humanretinas, providing a model system with whichto uncover the mechanisms of these develop-mental changes.

Thyroid hormone signaling and thetemporal switch between S and L/Mfate specification

Seminal work in mice identified Thrb2 as a crit-ical regulator of cone subtype specification: Thrb2mutants display a complete loss of M-opsin ex-pression and a complete gain of S-opsin expres-sion in cone photoreceptors (24–26). Similar rolesfor Thrb2 have been characterized in other or-ganisms with highly divergent cone patterning(27–29). Additionally, rare humanmutations inThrb2 are reported to alter color perception,which is indicative of a change in the S-to-L/Mcone ratio (30). To directly test the role of Thrb2in human cone subtype specification, we usedCRISPR/Cas9 in human embryonic stem cells(ESCs) to generate a homozygous mutation thatresulted in early translational termination in the

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1Department of Biology, Johns Hopkins University, 3400N. Charles Street, Baltimore, MD 21218, USA. 2Wilmer EyeInstitute, Johns Hopkins University School of Medicine,Baltimore, MD 21287, USA. 3Shiley Eye Institute, Universityof California, San Diego, La Jolla, CA 92093, USA. 4NationalInstitute of Mental Health, National Institutes of Health,Bethesda, MD 20892, USA. 5Department of ComputerScience, Johns Hopkins University, 3400 N. Charles Street,Baltimore, MD 21218, USA. 6Department of Molecular Biologyand Genetics, Johns Hopkins University School of Medicine,Baltimore, MD 21287, USA. 7Department of Neuroscience,Johns Hopkins University School of Medicine, Baltimore, MD21287, USA. 8Institute of Genetic Medicine, Johns HopkinsUniversity School of Medicine, Baltimore, MD 21287, USA.*Corresponding author. Email: robertjohnston@jhu.edu

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first exon of Thrb2 (fig. S2A). Surprisingly, organ-oids derived from these mutant stem cells dis-played no differences in cone subtype ratio fromgenotypically wild-type organoids [wild type, S =62%, L/M = 38%; Thrb2 knockout (KO), S = 59%,L/M = 41%; P = 0.83]. The S-to-L/M ratio is highfor both wild-type controls and Thrb2 KO organ-oids, likely owing to variability in organoid differ-entiation. Thus, unlike previous suggestions basedon other species, Thrb2 is dispensable for conesubtype specification in humans (Fig. 3, A to C).Because Thrb2 alone is not required for hu-

man cone subtype specification, we reexamineddata from Weiss et. al (30) and found that mis-sense mutations in exons 9 and 10 affected bothThrb2 and another isoform of the human Thrbgene, Thrb1 (fig. S2A). Thus, we asked whetherThrb1 and Thrb2 together are required for conesubtype specification in humans. To completelyablate Thrb function (Thrb1 and Thrb2), we usedCRISPR/Cas9 in human ESCs to delete a sharedexon that codes for part of the DNA bindingdomain of Thrb (fig. S2A). Thrb null mutantretinal organoids displayed a complete conver-sion of all cones to the S subtype (wild type, S =27%, L/M = 73%; Thrb KO, S = 100%, L/M = 0%;P < 0.0001) (Fig. 3, D to E and H). In these mu-tants, all cones expressed S-opsin and had the Scone morphology (Fig. 3, I and J). Thus, Thrb isrequired to activate L/M and to repress S conefates in the human retina.Thrb bindswith high affinity to triiodothyronine

(T3), themore active form of thyroid hormone, toregulate gene expression (31). Depletion or addition

of T3 alters the ratios of S to M cones in rodents(25, 32, 33). Because L/M cones differentiate afterS cones,wehypothesized that T3 acts throughThrblate in retinal development to induceL/Mcone fateand repress S cone fate. One prediction of this hy-pothesis is that addition of T3 early in develop-ment will induce L/M fate and repress S fate. Totest this model, we added 20 nM T3 to ESC- andiPSC-derived organoids starting from days 20 to50 and continued until day 200 of differentiation.We observed a dramatic conversion of cone cellsto L/M fate (wild type, S = 27%, L/M = 73%; wildtype + T3, S = 4%, L/M = 96%; P < 0.01) (Fig. 3, FandH, and fig. S2B). Thus, early addition of T3 issufficient to induce L/M fate and suppress S fate.To test whether T3 acts specifically through

Thrb to control cone subtype specification, wedifferentiated Thrb mutant organoids with earlyT3 addition. Thrb mutation completely suppressedthe effects of T3, generating organoids with onlyS cones (wild type + T3, S = 4%, L/M= 96%; ThrbKO+ T3, S = 100%, L/M= 0%; P < 0.0001) (Fig. 3,F to H).We conclude that T3 acts though Thrb topromote L/M cone fate and suppress S cone fate.We confirmed the regulation of L/M-opsin ex-

pression through thyroid hormone signaling in aretinoblastoma cell line, which expresses L/M-opsinwhen treatedwith T3 (fig. S2, C andD) (34).T3-induced activation of L/M-opsin expressionwassuppressed upon RNA interference knockdownof Thrb (fig. S2, E and F), which is similar to thesuppression observed in human organoids.In organoids, early T3 addition not only con-

verted cone cells to L/M fate but also dramatically

increased cone density (Fig. 3, F andK).Moreover,T3 acts specifically through Thrb to control conedensity (Fig. 3, G and K). Early T3 addition mayincrease cone density by advancing and extendingthe temporal window of L/M cone generation.Together, these results demonstrate that T3

signals though Thrb to promote L/M cone fateand repress S cone fate in developing humanretinal tissue.

Dynamic expression of thyroidhormone–regulating genesduring development

Our data suggest that temporal control of thyroidhormone signaling determines the S-versus-L/Mcone fate decision, in which low signaling earlyinduces S fate and high signaling late induces L/Mfate. Thyroid hormone exists largely in two states:thyroxine (T4), the most abundant circulatingform of thyroid hormone, and T3, which bindsthyroid hormone receptors with high affinity(31, 35). Because the culturemedium contains lowamounts of T3 and T4, we hypothesized that theretina itself could modulate and/or generate thy-roid hormone to control subtype fates.Conversion of T4 to T3 occurs locally in target

tissues to induce gene expression responses (36, 37).Deiodinases—enzymes that modulate the levelsof T3 andT4—are expressed in the retinas ofmice,fish, and chickens (29, 38–42). Therefore, we pre-dicted that T3- and T4-degrading enzymes wouldbe expressedduring early humaneyedevelopmentto reduce thyroid hormone signaling and specifyS cones, whereas T3-producing enzymes, carriers,

Eldred et al., Science 362, eaau6348 (2018) 12 October 2018 2 of 7

Fig. 1. S and L/M cone generation inhuman retinal organoids. (A) Decision be-tween S and L/M cone subtype fate.(B and C) S-opsin (blue) and L/M-opsin(green). (B) Human adult retina age 53.(C) iPSC-derived organoid, day 200 ofdifferentiation. (D to K) Bright-fieldimages of organoids derived from iPSCs.(D) Undifferentiated iPSCs. (E) Day 1, aggre-gation. (F) Day 4, formation of neuronalvesicles. (G) Day 8, differentiation of retinalvesicles. (H) Day 12, manual isolation ofretinal organoid. (I) Day 43, arrow indicatesdeveloping retinal tissue, and arrowheadindicates developing retinal pigment epithe-lium. (J) Day 199, arrow indicates outersegments. (K) Day 330, arrow indicates outersegments.

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and transporters would be expressed later in hu-man eye development to increase signaling andgenerate L/M cones.To test these predictions, we examined gene

expression across 250 days of organoid develop-ment. The expressionpatterns of thyroidhormone–regulating geneswere grouped into three classes:changing expression (Fig. 4A), consistent ex-pression (Fig. 4B), or no expression (Fig. 4C).Deiodinase 3 (DIO3), an enzyme that degradesT3 and T4 (36), was expressed at high levels earlyin organoid development but at low levels later(Fig. 4A). Conversely, deiodinase 2 (DIO2), an en-zyme that converts T4 to active T3 (36), was ex-pressed at low levels early but then dramaticallyincreased over time (Fig. 4A). We examined RNA-seq data from Hoshino et. al (23) and found thatdeveloping human retinas display similar tempo-ral changes in expression of DIO3 and DIO2(fig. S3A). Deiodinase 1 (DIO1), which regulatesT3 and T4 predominantly in the liver and kidney(43), was not expressed in organoids or retinas(Fig. 4C and fig. S3C). Thus, the dynamic expres-sion of Dio3 and Dio2 supports low thyroid hor-mone signaling early in development to generateS cones and high thyroid hormone signaling lateto produce L/M cones.Consistent with a role for high thyroid hor-

mone signaling in the generation of L/M coneslater in development, expression of transthyretin

(TTR), a thyroidhormone carrierprotein, increasedduring organoid and retinal development (Fig.4A and fig. S3A) (23). By contrast, albumin (ALB)and thyroxine-binding globulin (SERPINA7), othercarrier proteins of T3 and T4, were not expressedin organoids or retinas (Fig. 4C and fig. S3C) (23).T3 and T4 are transported into cells via mem-

brane transport proteins (44). The T3/T4 transport-ers SLC7A5 and SLC7A8 increased in expressionduring organoid differentiation (Fig. 4A). Addi-tionally, two T3/T4 transporters, SLC3A2 andSLC16A2, were expressed at high and consistentlevels throughout organoid development (Fig. 4B).Other T3/T4 transporters (SLC16A10, SLCO1C1,and SLC5A5) were not expressed in organoids(Fig. 4C), suggesting tissue-specific regulationof T3/T4 uptake. We observed similar expressionpatterns of T3/T4 transporters in human retinas(fig. S3, A to C) (23).We next examined expression of transcrip-

tional activators and repressors that mediate theresponse to thyroid hormone. Consistent withThrb expression in human cones (45), expressionof Thrb in organoids increased with time as conecells were specified (Fig. 4A). Expression of thyroidhormone receptor a (Thra) similarly increasedwith time (Fig. 4A). Thyroid hormone receptorcofactors, corepressor NCoR2 and coactivatorMED1, were expressed at steady levels duringorganoid differentiation (Fig. 4B). Similar tempo-

ral expression patterns were observed in humanretinas (fig. S3, A andB) (23). Thus, our data suggestthat expression of Thrb and other transcriptionalregulators enables gene regulatory responses todifferential thyroid hormone levels.A complex pathway controls production of thy-

roid hormone. Thyrotropin-releasing hormone(TRH) is produced by the hypothalamus andother neural tissue. TRH stimulates release ofthyroid-stimulating hormone a (CGA) and thyroid-stimulating hormone b (TSHb) from the pituitarygland. CGAandTSHb bind the thyroid-stimulatinghormone receptor (TSHR) in the thyroid gland.T3 andT4 production requires thyroglobulin (TG),the substrate for T3/T4 synthesis, and thyroid per-oxidase (TPO), an enzyme that iodinates tyrosineresidues in TG (46). TRH was expressed in organ-oids and retinas, but the other players were not(Fig. 4, A to C, and fig. S3, A to C) (23, 47, 48),suggesting that the retina itself does not gen-erate thyroid hormone; rather, it modulates therelative levels of T3 and T4 and expresses TRHto signal for thyroid hormone production in othertissues.Therefore, the temporal expression of thyroid

hormone signaling regulators supports ourmodelthat the retina intrinsically controls T3 and T4levels, ensuring low thyroid hormone signalingearly to promote S fate and high thyroid hormonesignaling late to specify L/M fate (Fig. 4D).

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Fig. 2. Human cone subtype specification isrecapitulated in organoids. (A to K) S-opsin(blue) and L/M-opsin (green) were examined inhuman iPSC-derived organoids [(A), (C) to (E),and (G) to (M)] and human retinas [(B), (D),(F), and (H)]. [(A) to (C) and (E) to (G)] Arrowsindicate outer segments, solid arrowheads indi-cate inner segments, and open arrowheadsindicate nuclei. [(A) and (E)] CRX (a generalmarker of photoreceptors) is expressed inS cones and L/M cones. [(B) to (D)] S conesdisplay short outer segments and thin innersegments in both human retinas and organoids.[(F) to (H)] L/M cones display long outersegments and wide inner segments in bothhuman retinas and organoids. [(D) and (H)]Quantification of outer segment lengths andinner segment widths (adult retina, L/M, n =13 cones, S, n = 10 cones; organoid, L/M, n =35 cones, S, n = 42 cones). [(I) to (N)] S conesare generated before L/M cones in organoids.(L) Ratio of S:L/M cones during organoid devel-opment. (M) Density of S and L/M cones duringorganoid development. (N) S-opsin expressionprecedes L/M-opsin expression in humaniPSC-derived organoids. CRX expression startsbefore opsin expression. TPM, transcripts perkilobase million.

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Organoids provide a powerful system with whichto determine the mechanisms of human develop-ment.Model organismandepidemiological studiesgenerate important hypotheses about human biol-ogy that are often experimentally intractable. Thiswork shows that organoids enable direct testingof hypotheses in developing human tissue.Our studies identify temporal regulation of

thyroid hormone signaling as a mechanism thatcontrols cone subtype specification in humans.Consistent with our findings, preterm human in-fants with low T3/T4 have an increased incidenceof color vision defects (49–52). Moreover, our iden-tification of amechanism that generates one conesubtypewhile suppressing the other, coupledwithsuccessful transplantation and incorporation ofstem cell–derived photoreceptors in mice (53–56),suggests that the promise of therapies to treathuman diseases such as color blindness, retinitispigmentosa, and macular degeneration will beachieved in the near future.

Materials and methods summaryCell lines

H7 ESC (WA07, WiCell) and episomal-derivedEP1.1 iPSC lines were used for differentiation.

WERI-Rb1 retinoblastoma cells were obtainedfrom ATCC. Cell maintenance and organoid dif-ferentiation protocols are described in the supple-mentary materials.

CRISPR mutations

All mutations were generated in H7 ESCs. Cellswere modified to express an inducible Cas9 ele-ment. Plasmids for guideRNA (gRNA) transfectionwere generated by using the pSpCas9(BB)-P2A-Puro plasmid modified from the pX459_V2.0plasmid (62988, Addgene) by replacing T2Awitha P2A sequence. Mutations were confirmed withpolymerase chain reaction sequencing. Genediagrams of deletions are displayed in fig. S2A.Detailed transfection procedures, gRNAsequences,and homology arm sequences are included in thesupplementary materials.

Immunohistochemistry

Primary antibodies were used at the followingdilutions: goat anti-SW-opsin (1:200 for orga-noids, 1:500 for human retinas) (Santa Cruz Bio-technology), rabbit anti-LW/MW-opsins (1:200 fororganoids, 1:500 for human retinas) (Millipore),mouse anti-CRX (1:500) (Abnova), and mouse

anti-Rhodopsin (1:500) (GeneTex). All secondaryantibodies were Alexa Fluor–conjugated (1:400)andmade in donkey (Molecular Probes). Detailedmethods for fixation, microscopy, and image pro-cessing of organoids, retinas, and WERI-Rb1 cellsare included in the supplementary materials.

Organoid ageOpsin expression time course

EP1 iPSC–derived organoids for time course ex-periments were binned into 10-day incrementsfor analysis. Organoids were binned into day 130[actual day 129 (n= 3 organoids)], day 150 [actualday 152 (n= 4 organoids)], day 170 [actual day 173(n = 2 organoids)], day 200 [actual days 194 to199 (n = 7 organoids)], day 290 [actual day 291(n = 3 organoids)], and day 360 [actual day 361(n = 3 organoids)]. Quantifications of outer-segment lengths and inner-segmentwidthsweremeasured in day 361 organoids (n = 3 organoids).

Opsin expression in different conditions

iCas9 H7 ESC–derived organoids for Thrb2 KOsand controls were analyzed at day 200. Organo-ids for Thrb KO, control, andwild-type + T3wereanalyzed at two time points: two organoids were

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Fig. 3. Thyroid hormone signaling is neces-sary and sufficient for the temporal switchbetween S and L/M fate specification. (A toK) S-opsin (blue) and L/M-opsin (green) wereexamined in human ESC-derived organoids.(A) Wild-type (WT). (B) Thrb2 early terminationmutant (Thrb2 KO). (C) Quantification of (A) and (B)(WT, n = 3 organoids; Thrb2 KO, n = 3 organoids).(D) WT. (E) Thrb KO. (F) WT treated with 20 nMT3 (WT + T3). (G) Thrb KO treated with 20 nMT3 (Thrb KO + T3). (H) Quantification of (D)to (G) (WT, n = 9 organoids; Thrb KO, n =3 organoids; WT + T3, n = 6 organoids; ThrbKO + T3, n = 3 organoids. Tukey’s multiplecomparisons test:WTversus Thrb KO, P < 0.0001;WT versus WT + T3, P < 0.01; WT + T3 versusThrb KO + T3, P < 0.0001). (I) Length of outersegments. WT, L/M n = 66 cells; WT, S n =66 cells; Thrb KO, n = 50 cells (Tukey’s multiplecomparisons test, WT L/M versus WT S,P < 0.0001; WT L/M versus Thrb KO, P < 0.0001;WT S versus Thrb KO, not significantly different).(J) Width of inner segments.WT, L/M n = 78 cells;WT, S n = 78 cells; Thrb KO, n = 118 cells(Tukey’s multiple comparisons test, WT L/Mversus WT S, P < 0.0001; WT L/M versus ThrbKO, P < 0.0001; WT S versus Thrb KO, notsignificantly different). (K) T3 acts through Thrbto increase total cone number. Quantificationof density of S and L/M cones; WT, n =6 organoids; Thrb KO, n = 3 organoids; WT + T3,n = 3 organoids; Thrb KO + T3, n = 3 organoids(Tukey’s multiple comparisons test betweentotal cone numbers, WT versus Thrb KO, notsignificantly different; WT versus WT + T3, P <0.01; WT + T3 versus Thrb KO + T3, P < 0.0001).

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taken at day 199 for each group, andonewas takenat day 277 for each group. T3-treated organoidswere taken at timepoints betweenday 195 andday200 for different differentiations. For each treat-ment group and genotype, organoids were com-pared with control organoids grown in parallel.

RNA-seq time course

EP1 iPSC–derived organoids were analyzed attime points ranging from day 10 to day 250 ofdifferentiation. We took samples at day 10 (n =3 organoids), day 20 (n = 2 organoids), day 35 (n =3 organoids), day 69 (n = 3 organoids), day 111(n=3organoids), day 128 (n=3organoids), day 158(n= 2 organoids), day 173 (n= 3 organoids), day 181

(n = 3 organoids), day 200 (n = 3 organoids), andday 250 (n = 3 organoids). RNA from individualorganoids was extracted by using the ZymoDirect-zol RNA Microprep Kit (Zymo Research)according to manufacturer’s instructions. Libra-ries were prepared by using the Illumina TruSeqstrandedmRNAkit and sequenced on an IlluminaNextSeq 500 with single 200–base pair reads.

RNA-seq time course analysis

Expression levels were quantified by using Kallisto(version 0.34.1) with the following parameters: “-b100 -l 200 -s 10 -t 20–single”. The Gencode release28 comprehensive annotation was used as thereference transcriptome (57). Transcripts per mil-

lion (TPM) values (table S1) were then used togenerate graphs in Prism and heatmaps in R byusing ggplot2. The distributions of transcriptswere plotted so as to identify the best low TPMcutoff (fig. S5A). The threshold was determinedto be 0.7 log(TPM + 1)—5 TPM—and this valuewas used as an inflection point for the heatmaps.Heatmaps for fig. S3, A to C, were made sim-ilarly, by using CPM values from Hoshino et. al(fig. S5B) (23).

Measurements and quantification

Measurements of retinal area and cell morphol-ogy were done by using ImageJ software. Quan-tifications and statistics (except for RNA-seq data)

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Fig. 4. Dynamic expression of thyroid hormone signaling regulators during development. (A to C) Heat maps of log(TPM + 1) values for genes with(A) changing expression, (B) consistent expression, and (C) no expression. Numbers at the bottom of heat maps indicate organoid age in days.(D) Model of the temporal mechanism of cone subtype specification in humans. For simplicity, only the roles of DIO3 and DIO2 are illustrated. In step 1,expression of DIO3 degrades T3 and T4, leading to S cone specification. In step 2, expression of DIO2 converts T4 to T3 to signal Thrb to repress Sand induce L/M cone fate.

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were done in GraphPad Prism, with a significancecutoff of 0.01. Statistical tests are listed in figurelegends. All error bars represent the SEM.

REFERENCES AND NOTES

1. K. Viets, K. Eldred, R. J. Johnston Jr., Mechanisms ofphotoreceptor patterning in vertebrates and invertebrates.Trends Genet. 32, 638–659 (2016). doi: 10.1016/j.tig.2016.07.004; pmid: 27615122

2. J. Nathans, D. Thomas, D. S. Hogness, Molecular genetics ofhuman color vision: The genes encoding blue, green, and redpigments. Science 232, 193–202 (1986). doi: 10.1126/science.2937147; pmid: 2937147

3. D. Vollrath, J. Nathans, R. W. Davis, Tandem array of humanvisual pigment genes at Xq28. Science 240, 1669–1672 (1988).doi: 10.1126/science.2837827; pmid: 2837827

4. Y. Wang et al., A locus control region adjacent to the humanred and green visual pigment genes. Neuron 9, 429–440(1992). doi: 10.1016/0896-6273(92)90181-C; pmid: 1524826

5. P. M. Smallwood, Y. Wang, J. Nathans, Role of a locus controlregion in the mutually exclusive expression of human red andgreen cone pigment genes. Proc. Natl. Acad. Sci. U.S.A. 99,1008–1011 (2002). doi: 10.1073/pnas.022629799;pmid: 11773636

6. Y. Wang et al., Mutually exclusive expression of human red andgreen visual pigment-reporter transgenes occurs at highfrequency in murine cone photoreceptors. Proc. Natl. Acad.Sci. U.S.A. 96, 5251–5256 (1999). doi: 10.1073/pnas.96.9.5251;pmid: 10220452

7. J. Nathans et al., Molecular genetics of human blue conemonochromacy. Science 245, 831–838 (1989). doi: 10.1126/science.2788922; pmid: 2788922

8. A. S. Ladekjaer-Mikkelsen, T. Rosenberg, A. L. Jørgensen,A new mechanism in blue cone monochromatism. Hum. Genet.98, 403–408 (1996). doi: 10.1007/s004390050229;pmid: 8792812

9. E. J. Patterson et al., Cone photoreceptor structure in patientswith X-linked cone dysfunction and red-green color visiondeficiency. Invest. Ophthalmol. Vis. Sci. 57, 3853–3863 (2016).doi: 10.1167/iovs.16-19608; pmid: 27447086

10. T. Nakano et al., Self-formation of optic cups and storablestratified neural retina from human ESCs. Cell Stem Cell 10,771–785 (2012). doi: 10.1016/j.stem.2012.05.009;pmid: 22704518

11. X. Zhong et al., Generation of three-dimensional retinal tissuewith functional photoreceptors from human iPSCs.Nat. Commun. 5, 4047 (2014). doi: 10.1038/ncomms5047;pmid: 24915161

12. K. J. Wahlin et al., Photoreceptor outer segment-like structuresin long-term 3D retinas from human pluripotent stem cells.Sci. Rep. 7, 766 (2017). doi: 10.1038/s41598-017-00774-9;pmid: 28396597

13. R. Kaewkhaw et al., Transcriptome dynamics of developingphotoreceptors in three-dimensional retina culturesrecapitulates temporal sequence of human cone and roddifferentiation revealing cell surface markers and genenetworks. Stem Cells 33, 3504–3518 (2015). doi: 10.1002/stem.2122; pmid: 26235913

14. M. J. Phillips et al., A novel approach to single cellRNA-sequence analysis facilitates in silico gene reporting ofhuman pluripotent stem cell-derived retinal cell types.Stem Cells 36, 313–324 (2018). doi: 10.1002/stem.2755;pmid: 29230913

15. A. Artero Castro, D. Lukovic, P. Jendelova, S. Erceg, Concisereview: Human induced pluripotent stem cell models of retinitispigmentosa. Stem Cells 36, 474–481 (2018). doi: 10.1002/stem.2783; pmid: 29345014

16. T. Furukawa, E. M. Morrow, C. L. Cepko, Crx, a novel otx-likehomeobox gene, shows photoreceptor-specific expressionand regulates photoreceptor differentiation. Cell 91, 531–541(1997). doi: 10.1016/S0092-8674(00)80439-0; pmid: 9390562

17. C. L. Freund et al., Cone-rod dystrophy due to mutations in anovel photoreceptor-specific homeobox gene (CRX)essential for maintenance of the photoreceptor. Cell 91,543–553 (1997). doi: 10.1016/S0092-8674(00)80440-7;pmid: 9390563

18. S. Chen et al., Crx, a novel Otx-like paired-homeodomainprotein, binds to and transactivates photoreceptor cell-specificgenes. Neuron 19, 1017–1030 (1997). doi: 10.1016/S0896-6273(00)80394-3; pmid: 9390516

19. C. A. Curcio et al., Distribution and morphology of human conephotoreceptors stained with anti-blue opsin. J. Comp. Neurol.

312, 610–624 (1991). doi: 10.1002/cne.903120411;pmid: 1722224

20. A. Hendrickson, D. Drucker, The development of parafoveal andmid-peripheral human retina. Behav. Brain Res. 49, 21–31(1992). doi: 10.1016/S0166-4328(05)80191-3; pmid: 1388798

21. M. Xiao, A. Hendrickson, Spatial and temporal expression ofshort, long/medium, or both opsins in human fetal cones.J. Comp. Neurol. 425, 545–559 (2000). doi: 10.1002/1096-9861(20001002)425:4<545::AID-CNE6>3.0.CO;2-3;pmid: 10975879

22. C. A. Curcio, K. R. Sloan, R. E. Kalina, A. E. Hendrickson,Human photoreceptor topography. J. Comp. Neurol. 292,497–523 (1990). doi: 10.1002/cne.902920402; pmid: 2324310

23. A. Hoshino et al., Molecular anatomy of the developing humanretina. Dev. Cell 43, 763–779.e4 (2017). doi: 10.1016/j.devcel.2017.10.029; pmid: 29233477

24. L. Ng et al., A thyroid hormone receptor that is required for thedevelopment of green cone photoreceptors. Nat. Genet. 27,94–98 (2001). doi: 10.1038/83829; pmid: 11138006

25. M. R. Roberts, M. Srinivas, D. Forrest, G. Morreale de Escobar,T. A. Reh, Making the gradient: Thyroid hormone regulatescone opsin expression in the developing mouse retina. Proc.Natl. Acad. Sci. U.S.A. 103, 6218–6223 (2006). doi: 10.1073/pnas.0509981103; pmid: 16606843

26. M. L. Applebury et al., Transient expression of thyroid hormonenuclear receptor TRbeta2 sets S opsin patterning during conephotoreceptor genesis. Dev. Dyn. 236, 1203–1212 (2007).doi: 10.1002/dvdy.21155; pmid: 17436273

27. S. C. Suzuki et al., Cone photoreceptor types in zebrafish aregenerated by symmetric terminal divisions of dedicatedprecursors. Proc. Natl. Acad. Sci. U.S.A. 110, 15109–15114(2013). doi: 10.1073/pnas.1303551110; pmid: 23980162

28. M. Sjöberg, B. Vennström, D. Forrest, Thyroid hormonereceptors in chick retinal development: Differential expressionof mRNAs for alpha and N-terminal variant beta receptors.Development 114, 39–47 (1992). pmid: 1576965

29. J. M. Trimarchi, S. Harpavat, N. A. Billings, C. L. Cepko, Thyroidhormone components are expressed in three sequentialwaves during development of the chick retina. BMC Dev. Biol.8, 101 (2008). doi: 10.1186/1471-213X-8-101; pmid: 18854032

30. A. H. Weiss, J. P. Kelly, D. Bisset, S. S. Deeb, Reduced L- andM- and increased S-cone functions in an infant with thyroidhormone resistance due to mutations in the THRb2 gene.Ophthalmic Genet. 33, 187–195 (2012). doi: 10.3109/13816810.2012.681096; pmid: 22551329

31. H. H. Samuels, J. S. Tsai, J. Casanova, F. Stanley, Thyroidhormone action: In vitro characterization of solubilizednuclear receptors from rat liver and cultured GH1 cells.J. Clin. Invest. 54, 853–865 (1974). doi: 10.1172/JCI107825;pmid: 4372251

32. A. Glaschke, M. Glösmann, L. Peichl, Developmental changes ofcone opsin expression but not retinal morphology in thehypothyroid Pax8 knockout mouse. Invest. Ophthalmol.Vis. Sci. 51, 1719–1727 (2010). doi: 10.1167/iovs.09-3592;pmid: 19834026

33. A. Glaschke et al., Thyroid hormone controls cone opsinexpression in the retina of adult rodents. J. Neurosci. 31,4844–4851 (2011). doi: 10.1523/JNEUROSCI.6181-10.2011;pmid: 21451022

34. Y. Liu, L. Fu, D. G. Chen, S. S. Deeb, Identification of novelretinal target genes of thyroid hormone in the human WERIcells by expression microarray analysis. Vision Res. 47,2314–2326 (2007). doi: 10.1016/j.visres.2007.04.023;pmid: 17655910

35. A. Schroeder, R. Jimenez, B. Young, M. L. Privalsky, The abilityof thyroid hormone receptors to sense t4 as an agonistdepends on receptor isoform and on cellular cofactors.Mol. Endocrinol. 28, 745–757 (2014). doi: 10.1210/me.2013-1335;pmid: 24673558

36. M. Dentice, A. Marsili, A. Zavacki, P. R. Larsen, D. Salvatore,The deiodinases and the control of intracellular thyroidhormone signaling during cellular differentiation. Biochim.Biophys. Acta 1830, 3937–3945 (2013). doi: 10.1016/j.bbagen.2012.05.007; pmid: 22634734

37. V. M. Darras, A. M. Houbrechts, S. L. Van Herck, Intracellularthyroid hormone metabolism as a local regulator of nuclearthyroid hormone receptor-mediated impact on vertebratedevelopment. Biochim. Biophys. Acta 1849, 130–141 (2015).doi: 10.1016/j.bbagrm.2014.05.004; pmid: 24844179

38. L. Ng et al., Type 3 deiodinase, a thyroid-hormone-inactivatingenzyme, controls survival and maturation of conephotoreceptors. J. Neurosci. 30, 3347–3357 (2010).doi: 10.1523/JNEUROSCI.5267-09.2010; pmid: 20203194

39. P. J. Bonezzi, M. E. Stabio, J. M. Renna, The development ofmid-wavelength photoresponsivity in the mouse retina.Curr. Eye Res. 43, 666–673 (2018). doi: 10.1080/02713683.2018.1433859; pmid: 29447486

40. E. Bagci et al., Deiodinase knockdown during early zebrafishdevelopment affects growth, development, energymetabolism, motility and phototransduction. PLOS ONE10, e0123285 (2015). doi: 10.1371/journal.pone.0123285;pmid: 25855985

41. C. Guo et al., Intrinsic expression of a multiexon type 3deiodinase gene controls zebrafish embryo size. Endocrinology155, 4069–4080 (2014). doi: 10.1210/en.2013-2029;pmid: 25004091

42. S. L. Bruhn, C. L. Cepko, Development of the pattern ofphotoreceptors in the chick retina. J. Neurosci. 16, 1430–1439(1996). doi: 10.1523/JNEUROSCI.16-04-01430.1996;pmid: 8778294

43. A. C. Bianco, D. Salvatore, B. Gereben, M. J. Berry,P. R. Larsen, Biochemistry, cellular and molecular biology,and physiological roles of the iodothyronine selenodeiodinases.Endocr. Rev. 23, 38–89 (2002). doi: 10.1210/edrv.23.1.0455;pmid: 11844744

44. D. S. Sharlin, T. J. Visser, D. Forrest, Developmental andcell-specific expression of thyroid hormone transporters in themouse cochlea. Endocrinology 152, 5053–5064 (2011).doi: 10.1210/en.2011-1372; pmid: 21878515

45. T. C. Lee, D. Almeida, N. Claros, D. H. Abramson, D. Cobrinik,Cell cycle-specific and cell type-specific expression of Rb inthe developing human retina. Invest. Ophthalmol. Vis. Sci. 47,5590–5598 (2006). doi: 10.1167/iovs.06-0063;pmid: 17122153

46. E. J. Barrett, in Medical Physiology, 2e Updated Edition,W. B. E. Boulpaep, Ed. (Elsevier, Inc., Philadelphia, PA, 2012),chap. 49.

47. S. R. Dubovy et al., Expression of hypothalamicneurohormones and their receptors in the human eye.Oncotarget 8, 66796–66814 (2017). doi: 10.18632/oncotarget.18358; pmid: 28977997

48. E. Martino et al., Thyrotropin-releasing hormone-like material inhuman retina. J. Endocrinol. Invest. 3, 267–271 (1980).doi: 10.1007/BF03348274; pmid: 6776180

49. J. Rovet, N. Simic, The role of transient hypothyroxinemia ofprematurity in development of visual abilities. Semin. Perinatol.32, 431–437 (2008). doi: 10.1053/j.semperi.2008.09.009;pmid: 19007682

50. N. Simic, C. Westall, E. V. Astzalos, J. Rovet, Visual abilities at6 months in preterm infants: Impact of thyroid hormonedeficiency and neonatal medical morbidity. Thyroid 20,309–315 (2010). doi: 10.1089/thy.2009.0128; pmid: 20144040

51. S. A. Yassin, A. J. Al-Dawood, W. M. Al-Zamil, M. A. Al-Ghamdi,Z. N. Al-Khudairy, Comparative study of visual dysfunctionsin 6-10-year-old very preterm- and full-term-born children.Int. Ophthalmol. (2018). doi: 10.1007/s10792-018-0959-2;pmid: 29916121

52. H. J. Dowdeswell, A. M. Slater, J. Broomhall, J. Tripp, Visualdeficits in children born at less than 32 weeks’ gestation withand without major ocular pathology and cerebral damage.Br. J. Ophthalmol. 79, 447–452 (1995). doi: 10.1136/bjo.79.5.447;pmid: 7612557

53. R. A. Pearson et al., Restoration of vision after transplantationof photoreceptors. Nature 485, 99–103 (2012). doi: 10.1038/nature10997; pmid: 22522934

54. A. O. Barnea-Cramer et al., Function of human pluripotent stemcell-derived photoreceptor progenitors in blind mice. Sci. Rep.6, 29784 (2016). doi: 10.1038/srep29784; pmid: 27405580

55. D. A. Lamba, J. Gust, T. A. Reh, Transplantation of humanembryonic stem cell-derived photoreceptors restores somevisual function in Crx-deficient mice. Cell Stem Cell 4, 73–79(2009). doi: 10.1016/j.stem.2008.10.015; pmid: 19128794

56. B. A. Tucker et al., Transplantation of adult mouse iPScell-derived photoreceptor precursors restores retinal structureand function in degenerative mice. PLOS ONE 6, e18992 (2011).doi: 10.1371/journal.pone.0018992; pmid: 21559507

57. J. Harrow et al., GENCODE: The reference human genomeannotation for The ENCODE Project. Genome Res. 22,1760–1774 (2012). doi: 10.1101/gr.135350.111; pmid: 22955987

ACKNOWLEDGMENTS

We thank A. Kolodkin, J. Nathans, and members of the Johnstonlaboratory for helpful comments on the manuscript. Funding:K.C.E. was a Howard Hughes Medical Institute Gilliam Fellow andwas supported by the National Science Foundation GraduateResearch Fellowship Program under grant 1746891. R.J.J. was

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supported by the Pew Scholar Award 00027373. AuthorContributions: K.C.E.: Conception, data acquisition, newreagent contribution, data analysis, and data interpretation;drafted and revised manuscript. S.E.H.: Data acquisition anddata interpretation. K.A.H.: Data acquisition, data analysis, anddata interpretation. B.B.: Data analysis and data interpretation.P.-W.Z.: New reagent contribution. X.C.: New reagentcontribution. V.M.S.: New reagent contribution. D.S.W.: Newreagent contribution. S.H.: Data interpretation. J.T.: Data

analysis and data interpretation. K.W.: Data acquisition and newreagent contribution. D.J.Z.: Data acquisition and new reagentcontribution. R.J.J.: Conception and data interpretation;drafted and revised manuscript. Competing interests: None.Data and materials availability: RNA-seq data are available onGene Expression Omnibus, accession no. GSE119320. All otherdata and methods are in the supplementary materials. H7 stemcells are available from WiCell under a materials transferagreement with WiCell.

SUPPLEMENTARY MATERIALS

www.sciencemag.org/content/362/6411/eaau6348/suppl/DC1Materials and MethodsFigs. S1 to S5Table S1References (58–60)

30 June 2018; accepted 30 August 201810.1126/science.aau6348

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Thyroid hormone signaling specifies cone subtypes in human retinal organoids

Sluch, Derek S. Welsbie, Samer Hattar, James Taylor, Karl Wahlin, Donald J. Zack and Robert J. Johnston Jr.Kiara C. Eldred, Sarah E. Hadyniak, Katarzyna A. Hussey, Boris Brenerman, Ping-Wu Zhang, Xitiz Chamling, Valentin M.

DOI: 10.1126/science.aau6348 (6411), eaau6348.362Science 

, this issue p. eaau6348Sciencehormone receptor.later. The switch toward development of L/M cones depended on thyroid hormone signaling through the nuclear thyroid short-wavelength (S) opsin developed first, and cones expressing long- and medium-wavelength (L/M) opsin developedfound that differentiation of cone cells into their tuned subtypes was regulated by thyroid hormone. Cones expressing

studied organoids that recapitulate the development of the human retina andet al.opsin pigments they express. Eldred Cone photoreceptors in the eye enable color vision, responding to different wavelengths of light according to what

Thyroid hormone in color vision development

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